Principle of Assay
Rat Estradiol ELISA Kit (A327153) employs the competitive enzyme immunoassay technique for the quantitative measurement of rat Estradiol in serum, plasma, urine, and saliva. The 96-well microtiter plate has been pre-coated with Estradiol antigen. During the incubation, Estradiol present in the samples or standards competes with the fixed amount of immobilized Estradiol for binding sites on the Biotinylated Anti-Estradiol Antibody. The more Estradiol present in a sample or standard, the less Biotinylated Anti-Estradiol Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Estradiol Antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is inversely proportional to the amount of Estradiol present in each sample or standard. The concentration of Estradiol can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
